This invention relates generally to genetic package display (e.g., phage display), and in particular, to selection of ligands that bind to a cell surface receptor and internalize useful in gene therapy, screening, and in various methodologies. The methods described herein are also referred to as Ligand Identification Via Expression or xe2x80x9cLIVE(trademark)xe2x80x9d.
Bacteriophage expressing a peptide on its surface has been used to identify protein binding domains, including antigenic determinants, antibodies that are specifically reactive, mutants with high affinity binding, identify novel ligands, and substrate sites for enzymes. In its most common form, a peptide is expressed as a fusion protein with a coat protein of a filamentous phage. This results in the display of the foreign protein on the surface of the phage particle. Libraries of phages are generated that express a multitude of foreign proteins. These libraries are bound to a substrate or cell that presents the binding partner of interest. This screening process is essentially an affinity purification. Bound phage are recovered, propagated, and the gene encoding the foreign protein may be isolated and characterized. This technology is commonly referred to as xe2x80x9cphage display.xe2x80x9d
Through a process called xe2x80x9cbiopanning,xe2x80x9d the specific phage carrying a peptide or protein that interacts with a protein or other moiety on a solid phase can be identified and isolated. However, in many applications, binding or binding affinity is not the sole critical parameter. For example, in gene therapy, a gene sequence needs to be introduced into a cell. In preferred methods, the gene sequence is targeted to particular cells by way of a ligand/cell surface receptor interaction. Thus, the ligand must not only bind to the cells but must also be internalized and lead to expression of the introduced nucleic acid sequence. A native ligand that is internalized, when used in a system for gene therapy may not be efficiently internalized or while internalized may not lead to gene expression. For example, both FGF2 and EGF are internalizing ligands. Further, while many ligands can be found to internalize many do not facilitate tranduction of the targeted cell, leaving the internalized nucleic acid sequence in a non-functional state.
Phage libraries can be screened for potentially internalizing ligands by biopanning on live cells and rescuing internalized phage from the cells after stripping off externally bound phage (e.g., acid elution). However, this method may result in recovery of undesired phage that bind very tightly or are only partially internalized. Moreover, phage that are internalized and subjected to proteases lose infectivity and can not be recovered.
Generally speaking, the selection of ligands from phage display libraries or other genetic packages relies on peptide affinity and avidity. The number of phage recovered is determined by the complexity of the library, the target protein, and the selection stringency. Accordingly, prior to the present invention three types of selection (shown in FIG. 1) have been evaluated over the last several years: 1) affinity selection against simple targets like immobilized proteins; 2) affinity selection against complex targets such as the cell surface; and 3) selection after phage processing such as their internalization by cells.
When utilizing affinity selection, unbound phage are washed away with buffers of different stringencies and the remaining attached phage particles are recovered, amplified in bacteria, and then further enriched by repeated rounds of adsorption and recovery. In early rounds of selection, specific binding phage may be present among millions, if not billions, of other phage particles depending on the complexity of the library. While the phage recovered may be present in extremely low concentrations, they must be recovered in an infective form in order to allow for amplification by infecting host bacteria. As the selection is repeated, the library is significantly reduced in complexity and phage encoding the binding ligands can then be characterized by DNA sequencing.
With the success of ligand selection using phage libraries screened against immobilized proteins, investigators next began to select against whole cells Hoogenboom et al., European J. of Biochem. 260:774-784, 1999; Szardenings et al., J. Biol. Chem. 272(44):27943-27948, 1997; Pereira et al., J. Immunol Meth 203(1):11-24, 1997; Pasqualini et al., Nature 380:364-366, 1996. A clear advantage of this kind of xe2x80x9cbiopanningxe2x80x9d is that little or no prior knowledge of the target molecule (i.e. a receptor) is needed and it can be in its native form on the cell surface. But the fact that the target protein may be low in concentration relative to the other cell surface proteins presents a significant disadvantage to selection and as in affinity selection against immobilized targets, non-specifically adherent phage can give false positive signals. A low concentration of non-specific phage can interfere in the early rounds of selection when the true binders are in extremely low concentrations.
Despite these issues, the selection of peptides on complex targets has been successful. Recent studies by Pasqualini""s laboratory, have extended this approach even further by demonstrating that organ homing peptides can be selected from libraries that are xe2x80x9cbiopannedxe2x80x9d in-vivo. By applying standard phage display selection techniques to mice, in-vivo, they identified peptides capable of selectively targeting phage to the vasculature of different organs including, brain, prostate, and kidney. Pasqualini and Ruoslahti, Mol. Psychiatry 1(6):423, 1996.
In an effort to increase selection stringency and overcome the problems of non-specific adsorption that are associated with biopanning against whole cells, alternative strategies have been explored. Hart et al. initially demonstrated that RGD targeted phage are internalized through receptor mediated endocytosis. J. Biol. Chem. 269(17):12468-12474, 1994 Subsequently, Barry et al. showed that cell-specific internalizing peptides can be selected from large diverse libraries of displayed peptides by washing phage off the cell surface at low pH and recovering internalized phage from cell lysates. Nat. Med. 2:299-305, 1996. However, these methods suffered from multiple steps as well as having no clear ability to determine in an initial screen which ligands would facilitate gene transduction and which would not. The original rationale behind selection by internalization was merely to increase the stringency of selection and therefore increase the ratio of signal to background.
Accordingly, current methodologies are inadequate to determine the usefulness of ligands for facilitating transfer and transduction of a cell by a nucleic acid molecule associated with the genetic package and ligand.
Further, identification of target cells or tissues that are able to internalize ligands and express a transgene would readily allow one to identify specific target cells for known or putative ligands as well as allow one to identify ligands for specific cell or tissue types. However, current methods of target cell identification are hampered by the same difficulties, as noted above, with regard to screening for internalizing ligands. Accordingly, current methodologies are inadequate to determine which cell or tissue types are useful targets for ligand mediated gene transfer.
Thus, current screening methods are inadequate for selecting peptide or protein ligands that bind to a cell surface receptor, internalize and lead to expression of the carried nucleic acid molecule. The present invention discloses display methods that select peptide or protein ligands that internalize and facilitate cell transduction and expression of product from an associated nucleic acid molecule, and further provides other related advantages.
The present invention utilized novel genetic package display of putative ligands to investigate the ability of these molecules to facilate cellular transduction with associated nucleic acid molecules, while also providing a functional genomic benefit, in that a variety of sequences can be screened for their ability to successfully deliver a reporter gene or other nucleic acid molecule to a cell by analyzing expression of that nucleic acid molecule. The finding that this could be achieved is quite surprising as many genetic packages, including filamentous phages were thought to be too large to pass through an endosomal pathway and even if they did, it seemed unlikely that a bacterial virus could traffic appropriately through the endosomal environment, uncoat and express their single-stranded DNA in a mammalian cell. Barry et al. (supra) Remarkably, however, as demonstrated herein, significant levels of gene transfer are obtained when phage and are targeted to mammalian cells.
In principle, the genetic selection of functional ligands by the methods set forth herein represents a significant departure from traditional biopanning (see Table 1) because it increases the stringency of selection by requiring the displayed ligand to bind, internalize, taffick to the desired cellular location and deliver a selectable genetic marker. This increased stringency decreases background from phage displaying simple binding proteins. In addition, biopanning relies on the recovery of infective phage, whereas, selection by the methods described herein does not require the presence of infective phage. Therefore, the present methods allow one to recover phage that are subjected to proteolytic cleavage after internalization that would otherwise be lost during biopanning. Moreover, because selection used in the present invention is genetic, a stable inherited change in the cell (e.g., marker expression) can be used as the basis for selection. Thus, for example, it is feasible that stable cell colonies could be used to directly identify rare phage internalization events in one round of screening.
Accordingly, through the use of the present invention one of ordinary skill in the art could functionally assess a variety of displayed peptides, polypeptides, etc. for the ability to facilitate internaliztion and genetic transduction. Thus, a logical extension of this methodology is the use of these methods to functionally explore the existence of natural ligands present in existing libraries, such as those now deposited due to the human genome project. In this case, it is the cell surface, itself, that selects the xe2x80x9cmost fitxe2x80x9d ligand by their ability to stimulate receptor mediated endocytosis and subsequent phage transduction. The identification of novel ligands and their receptors using the present methodologies is likely to lead to new drugs and drug targets because cell-surface interacting ligands effect critical cellular processes like cell growth and differentiation. After all, natural ligands having direct clinical utility are the leading therapeutic products in biotechnology (e.g., erythropoietin, growth hormone, IL-2, GM-CSF). Yet, such ligands are the most difficult to mine from published genomic databases because they often exist as small fragments contained in much larger genes that are processed in a cell specific fashion.
Further, the present invention lends itself to the discovery of ligands useful for more traditional therapeutics. For example, once a sequence is identified that facilitates genetic transduction this ligand could be used to target small molecules (e.g., pharmaceutical drugs) to the nucleus of a cell or to other xe2x80x9ctargetedxe2x80x9d areas within a cell thus increasing the therapeutic efficacy of the associated drug.
Within one aspect of the present invention, a method of selecting internalizing ligands displayed on a genetic package is presented, comprising: (a) contacting a ligand displaying genetic package(s) with a cell(s), wherein the package carries a gene encoding a detectable product which is expressed upon internalization of the package; and (b) detecting product expressed by the cell(s); thereby selecting internalizing ligands displayed on a genetic package.
In another aspect, the invention provides a method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a selectable marker which is expressed upon internalization of the package, (b) detecting the selectable marker expressed by the cell(s); and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) expressing the product, and thereby identifying an internalizing ligand displayed on a genetic package.
In yet another aspect, the invention provides a method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a selectable product which is expressed upon internalization of the package, (b) incubating the cell(s) under selective conditions; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) which grow under the selective conditions; thereby identifying an internalizing ligand displayed on a genetic package.
In yet another aspect, a method is provided for a high throughput method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s) in an array, wherein each package carries a gene encoding at least one detectable product which is expressed upon internalization of the package; and (b) detecting product(s) expressed by the cell(s) in the array, and thereby identifying an internalizing ligand displayed on a genetic package. In one embodiment, the ligand displaying package comprises a library of ligand displaying packages.
In another aspect, the present invention provides a method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying a genetic packages with a cell(s), wherein each package carries a selectable marker which is detectable upon internalization of the package, (b) detecting the selectable marker internalized by the cells; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) carrying the selectable marker, thereby identifying an internalizing ligand displayed on a genetic package.
Within one aspect of the present invention, a method of selecting internalizing ligand/anti-ligand pairs is presented, comprising: (a) contacting a ligand displaying genetic package(s) with a cell(s), wherein the package carries a gene encoding a detectable product which is expressed upon internalization of the package; and (b) detecting product expressed by the cell(s); thereby selecting ligand/anti-ligand pairs.
In another aspect, the invention provides a method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand pair, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface; (b) detecting product expressed by the cell(s); and (c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) expressing the product, and thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand pair.
In yet another aspect, the invention provides a method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand pair, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface; (b) incubating the cell(s) under selective conditions; and (c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) which grow under the selective conditions; thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand pair.
In yet another aspect, a method is provided for a high throughput method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand interactions, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s) in an array, wherein each package carries a gene encoding at least one detectable product which is expressed upon internalization of the package; and (b) detecting product(s) expressed by the cell(s) in the array, and thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand interactions. In one embodiment, the array contains cells expressing a library of anti-ligand-receptor fusion proteins. In another embodiment, the ligand displaying package comprises a library of ligand displaying packages.
Within one aspect of the present invention, a method of identifying a target cell or tissue for internalizing ligands is presented, comprising: (a) contacting a library of ligand displaying genetic packages with a cell(s) or tissue(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package; and (b) detecting product expressed by the cell(s) or tissue(s), and thereby identifying a target cell or tissue for internalizing ligands.
In another aspect, the invention provides a method of selecting an internalizing ligand for a selected target cell or tissue within a pool of target cells or tissues and identifying a target cell or tissue for the internalizing ligand, comprising: (a) contacting a library of ligand displaying genetic packages with a pool of cell(s) or tissue(s), wherein each package carries a gene encoding a selectable marker which is expressed upon internalization of the package; (b) detecting the selectable marker expressed by the cell(s) or tissue(s); and (c) recovering a nucleic acid molecule encoding an internalizing ligand from a selected set of cell(s) or tissue(s) within the pool expressing the product.
In yet another aspect, the invention provides a method of selecting an internalizing ligand for a selected target cell or tissue within a pool of target cells or tissues and identifying a target cell or tissue for the internalizing ligand, comprising: (a) contacting a library of ligand displaying genetic packages with a pool of cell(s) or tissue(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package; (b) incubating the cell(s) or tissue(s) under selective conditions; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from a selected set of cell(s) or tissue(s) within the pool which grow under the selective conditions; thereby selecting internalizing ligands and identifying a target cell or tissue for the internalizing ligand.
In yet another aspect, a method is provided for a high throughput method of identifying target cells or tissues for internalizing ligands, comprising: (a) contacting a library of ligand displaying genetic packages with cells or tissue in an array, wherein each package carries a gene encoding at least one detectable product which is expressed upon internalization of the package; and (b) detecting product(s) expressed by the cells or tissue in the array; thereby identifying target cells or tissues for internalizing ligands. In one embodiment, the array contains a variety of cell types. In another embodiment, the method further comprises step (c), wherein the library is a library of ligand displaying bacteriophages that is repeatedly divided into subset pools and screened using steps (a) and (b) until a specific bacteriophage expressing an internalizing ligand is identified.
In yet additional embodiments a medicament for gene therapy is provided, comprising an internalizing ligand identified by the of the present invention. Also provided are anti-bacterial agents comprising an internalizing ligand identified by the methods of the present invention.
Also provided are methods for identifying transduction facilitating peptides, comprising: (a) contacting one or more ligand displaying a genetic packages with a cell(s), wherein each package displays a putative transduction facilitating peptide and a ligand known to internalize, and wherein each package carries a selectable marker which is detectable upon internalization of the package, (b) detecting the selectable marker internalized by the cells; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) carrying the selectable marker, and thereby identifying an internalizing ligand displayed on a genetic package.
In related embodiments, the selectable marker is selected from reporter gene expression, expression of a gene that confers the ability to permit cell growth under selection conditions, non-endogenous nucleic acid sequences that permit PCR amplification, and nucleic acid sequences that can be purified by protein/DNA binding.
In preferred embodiments, the ligand displaying genetic package comprises a bacteriophage. The bacteriophage are filamentous phage or lambdoid phage in other preferred embodiments. In some embodiments, the bacteriophage carries a genome vector. In other embodiments, the bacteriophage carries a hybrid vector.
In other embodiments, the library is a cDNA library, an antibody gene library, a random peptide gene library, or a mutein library. In other preferred embodiments, the detectable product is selected from the group consisting of green fluorescent protein, xcex2-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, human growth hormone and neomycin phosphotransferase.
In other embodiments, the cells may be isolated by flow cytometry, for example. In further embodiments, the methods further comprise recovering a nucleic acid molecule encoding the ligand from the cell(s) expressing the product. Also provided are methods for enhancing transduction by utilizing genotoxic agents, heat shock, and transduction facilitating peptides.
In certain embodiments, PCR or Hirt extraction methods are used to recover the internalized nucleic acid molecules.